Glycosyl transferase activity of the Escherichia coli penicillin-binding protein 1b: specificity profile for the substrate

The glycosyl transferase of the Escherichia coli bifunctional penicillin-binding protein (PBP) 1b catalyzes the assembly of lipid-transported N-acetylglucosaminyl-beta-1,4-N-acetylmuramoyl-L-Ala-gamma-D-Glu-meso-A2pm-D-Ala-D-Ala units (lipid II) into linear peptidoglycan chains. These units are linked, at C1 of N-acetylmuramic acid (MurNAc), to a C55 undecaprenyl pyrophosphate. In an in vitro assay, lipid II functions both as a glycosyl donor and as a glycosyl acceptor substrate. Using substrate analogues, it is suggested that the specificity of the enzyme for the glycosyl donor substrate differs from that for the acceptor. The donor substrate requires the presence of both N-acetylglucosamine (GlcNAc) and MurNAc and a reactive group on C1 of the MurNAc and does not absolutely require the lipid chain which can be replaced by uridine. The enzyme appears to prefer an acceptor substrate containing a polyprenyl pyrophosphate on C1 of the MurNAc sugar. The problem of glycan chain elongation that presumably proceeds by the repetitive addition of disaccharide peptide units at their reducing end is discussed.     

Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP1B, in Escherichia coli

The murein (peptidoglycan) sacculus is an essential polymer embedded in the bacterial envelope. The Escherichia coli class B penicillin-binding protein (PBP) 3 is a murein transpeptidase and essential for cell division. In an affinity chromatography experiment, the bifunctional transglycosylase-transpeptidase murein synthase PBP1B was retained by PBP3-sepharose when a membrane fraction of E. coli was applied. The direct protein-protein interaction between purified PBP3 and PBP1B was characterized in vitro by surface plasmon resonance. The interaction was confirmed in vivo employing two different methods: by a bacterial two-hybrid system, and by cross-linking/co-immunoprecipitation. In the bacterial two-hybrid system, a truncated PBP3 comprising the N-terminal 56 amino acids interacted with PBP1B. Both synthases could be cross-linked in vivo in wild-type cells and in cells lacking FtsW or FtsN. PBP1B localized diffusely and in foci at the septation site and also at the side wall. Statistical analysis of the immunofluorescence signals revealed that the localization of PBP1B at the septation site depended on the physical presence of PBP3, but not on the activity of PBP3. These studies have demonstrated, for the first time, a direct interaction between a class B PBP (PBP3) and a class A PBP (PBP1B) in vitro and in vivo, indicating that different murein synthases might act in concert to enlarge the murein sacculus during cell division.     

In vitro murein peptidoglycan synthesis by dimers of the bifunctional transglycosylase-transpeptidase PBP1B from Escherichia coli

PBP1B is a major bifunctional murein (peptidoglycan) synthase catalyzing transglycosylation and transpeptidation reactions in Escherichia coli. PBP1B has been shown to form dimers in vivo. The K(D) value for PBP1B dimerization was determined by surface plasmon resonance. The effect of the dimerization of PBP1B on its activities was studied with a newly developed in vitro murein synthesis assay with radioactively labeled lipid II precursor as substrate. Under conditions at which PBP1B dimerizes, the enzyme synthesized murein with long glycan strands (>25 disaccharide units) and with almost 50% of the peptides being part of cross-links. PBP1B was also capable of synthesizing trimeric muropeptide structures. Tri-, tetra-, and pentapeptide compounds could serve as acceptors in the PBP1B-catalyzed transpeptidation reaction.     

Effects of lemongrass oil on the morphological characteristics and peptidoglycan synthesis of Escherichia coli cells

The antibacterial effect of lemongrass oil, obtained from the aerial part of Cymbopogon citratus, on cells of Escherichia coli was investigated by electron microscopy and by measuring cell wall formation. Two strains of E. coli K-12 were used, one of which required diaminopimelic acid in the growth medium for its murein formation. Lemongrass oil was found to elicit morphological changes like filamentation, inhibition of septum formation, spheroplast formation, production of 'blisters', 'bulges' or mesosomes, as well as lysis and development of abnormally shaped cells. The incorporation of radioactively labelled diaminopimelic acid into the cell wall murein of strain W7, was inhibited by lemongrass oil in a dose dependent way. The sequence of changes induced by lemongrass oil on bacterial cell morphology and also its interference with murein synthesis in E. coli cells were interpreted to involve the penicillin binding proteins PBP 2 and PBP 3.     

Crystal structure of penicillin-binding protein 1a (PBP1a) reveals a mutational hotspot implicated in beta-lactam resistance in Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen whose infections have been treated with beta-lactam antibiotics for over 60 years, but the proliferation of strains that are highly resistant to such drugs is a problem of worldwide concern. Beta-lactams target penicillin-binding proteins (PBPs), membrane-associated enzymes that play essential roles in the peptidoglycan biosynthetic process. Bifunctional PBPs catalyze both the polymerization of glycan chains (glycosyltransfer) and the cross-linking of adjacent pentapeptides (transpeptidation), while monofunctional enzymes catalyze only the latter reaction. Although S. pneumoniae has six PBPs, only three (PBP1a, PBP2x, PBP2b) are major resistance determinants, with PBP1a being the only bifunctional enzyme. PBP1a plays a key role in septum formation during the cell division cycle and its modification is essential for the development of high-level resistance to penicillins and cephalosporins. The crystal structure of a soluble form of pneumococcal PBP1a (PBP1a*) has been solved to 2.6A and reveals that it folds into three domains. The N terminus contains a peptide from the glycosyltransfer domain bound to an interdomain linker region, followed by a central, transpeptidase domain, and a small C-terminal unit. An analysis of PBP1a sequences from drug-resistant clinical strains in light of the structure reveals the existence of a mutational hotspot at the entrance of the catalytic cleft that leads to the modification of the polarity and accessibility of the mutated PBP1a active site. The presence of this hotspot in all variants sequenced to date is of key relevance for the development of novel antibiotherapies for the treatment of beta-lactam-resistant pneumococcal strains.     

The glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae catalyzes the polymerization of murein glycan chains

The bacterial peptidoglycan consists of glycan chains of repeating beta-1,4-linked N-acetylglucosaminyl-N-acetylmuramyl units cross-linked through short peptide chains. The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs). The beta-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria. In this context, the GT catalytic domain represents a potential target in the antibacterial fight. In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant Streptococcus pneumoniae PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented. Dansylated lipid II was used as the substrate, and the kinetic parameters K(m) and k(cat)/K(m) were measured at 40.6 micro M (+/- 15.5) and 1 x 10(-3) M(-1) s(-1), respectively. The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin. Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding. In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor. The characterization of the catalytically active GT domain of S. pneumoniae PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal.     

The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan-polymerizing penicillin-binding protein 1b of Escherichia coli

The penicillin-binding protein (PBP) 1b of Escherichia coli catalyses the assembly of lipid-transported N-acetyl glucosaminyl-beta-1, 4-N-acetylmuramoyl-L-alanyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl+ ++- (L)-D-alanyl-D-alanine disaccharide pentapeptide units into polymeric peptidoglycan. These units are phosphodiester linked, at C1 of muramic acid, to a C55 undecaprenyl carrier. PBP1b has been purified in the form of His tag (M46-N844) PBP1bgamma. This derivative provides the host cell in which it is produced with a functional wall peptidoglycan. His tag (M46-N844) PBP1bgamma possesses an amino-terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an congruent with 100-amino-acid insert, to a D198-G435 glycosyl transferase module that possesses the five motifs characteristic of the PBPs of class A. In in vitro assays, the glycosyl transferase of the PBP catalyses the synthesis of linear glycan chains from the lipid carrier with an efficiency of congruent with 39 000 M-1 s-1. Glu-233, of motif 1, is central to the catalysed reaction. It is proposed that the Glu-233 gamma-COOH donates its proton to the oxygen atom of the scissile phosphoester bond of the lipid carrier, leading to the formation of an oxocarbonium cation, which then undergoes attack by the 4-OH group of a nucleophile N-acetylglucosamine. Asp-234 of motif 1 or Glu-290 of motif 3 could be involved in the stabilization of the oxocarbonium cation and the activation of the 4-OH group of the N-acetylglucosamine. In turn, Tyr-310 of motif 4 is an important component of the amino acid sequence-folding information. The glycosyl transferase module of PBP1b, the lysozymes and the lytic transglycosylase Slt70 have much the same catalytic machinery. They might be members of the same superfamily. The glycosyl transferase module is linked, via a short junction site, to the amino end of a Q447-N844 acyl transferase module, which possesses the catalytic centre-defining motifs of the penicilloyl serine transferases superfamily. In in vitro assays with the lipid precursor and in the presence of penicillin at concentrations sufficient to derivatize the active-site serine 510 of the acyl transferase, the rate of glycan chain synthesis is unmodified, showing that the functioning of the glycosyl transferase is acyl transferase independent. In the absence of penicillin, the products of the Ser-510-assisted double-proton shuttle are glycan strands substituted by cross-linked tetrapeptide-pentapeptide and tetrapeptide-tetrapeptide dimers and uncross-linked pentapeptide and tetrapeptide monomers. The acyl transferase of the PBP also catalyses aminolysis and hydrolysis of properly structured thiolesters, but it lacks activity on D-alanyl-D-alanine-terminated peptides. This substrate specificity suggests that carbonyl donor activity requires the attachment of the pentapeptides to the glycan chains made by the glycosyl transferase, and it implies that one and the same PBP molecule catalyses transglycosylation and peptide cross-linking in a sequential manner. Attempts to produce truncated forms of the PBP lead to the conclusion that the multimodular polypeptide chain behaves as an integrated folding entity during PBP1b biogenesis.     

Interaction of monoclonal antibodies with the enzymatic domains of penicillin-binding protein 1b of Escherichia coli.

Monoclonal antibodies (MAbs) against four different antigenic determinants of penicillin-binding protein (PBP) 1b were used to study the transglycosylase and transpeptidase activities of PBP 1b. Enzyme kinetics in the presence of and without the MAbs were determined, and the synthesized murein was analyzed. Two MAbs against the transglycosylase domain of PBP 1b appeared to inhibit this reaction. One MAb inhibited only the transpeptidase reaction, and one inhibited both enzymatic activities of PBP 1b. The latter two MAbs bound to the transpeptidase domain of PBP 1b. The following major conclusions were deduced from the results. (i) Transpeptidation is the rate-limiting step of the reaction cascade, and it is dependent on the product of transglycosylation. (ii) PBP 1b has only one type of transpeptidase activity, i.e., a penta-tetra transpeptidase activity. (iii) PBP 1b is probably a globular protein which has two intimately associated enzymatic domains.     

The essential cell division protein FtsN interacts with the murein (peptidoglycan) synthase PBP1B in Escherichia coli

Bacterial cell division requires the coordinated action of cell division proteins and murein (peptidoglycan) synthases. Interactions involving the essential cell division protein FtsN and murein synthases were studied by affinity chromatography with membrane fraction. The murein synthases PBP1A, PBP1B, and PBP3 had an affinity to immobilized FtsN. FtsN and PBP3, but not PBP1A, showed an affinity to immobilized PBP1B. The direct interaction between FtsN and PBP1B was confirmed by pulldown experiments and surface plasmon resonance. The interaction was also detected by bacterial two-hybrid analysis. FtsN and PBP1B could be cross-linked in intact cells of the wild type and in cells depleted of PBP3 or FtsW. FtsN stimulated the in vitro murein synthesis activities of PBP1B. Thus, FtsN could have a role in controlling or modulating the activity of PBP1B during cell division in Escherichia coli.     

Cell shape determination in Escherichia coli

The rigid cell wall peptidoglycan (murein) is a single giant macromolecule whose shape determines the shape of the bacterial cell. Insight into morphogenetic mechanism(s) responsible for determining the shape of the murein sacculus itself has begun to emerge only in recent years. The discovery that MfreB and Mbl are cytoskeletal actin homologues that form helical structures extending from pole to pole in rod-shaped cells has opened an exciting new field of microbial cell biology. MreB (in Gram-negative rods) and Mbl (in Gram-positive species) are essential for murein synthesis along the lateral wall and hence, the rod shape of the cell. Known members of the morphogenetic system include MreB (or Mbl), MreC, MreD and PBP2, but Rod A and murein biosynthetic enzymes involved in peptidoglycan precursor synthesis and assembly are likely to be recruited to the same multimolecular apparatus. However, the actual role of MreB in assembly of the morphogenetic complex is still not clear and little is known about regulatory mechanisms controlling the switch from lateral murein elongation to septa1 murein synthesis at the time of cell division.     

Penicillin-binding proteins 1a and 1b form independent dimers in Escherichia coli

We report here that PBP1a can dimerize but does not interact with PBP1b to form PBP1a/PBP1b heterodimers in Escherichia coli. These findings support the idea of a relevant involvement of dimerization of both PBP1a and PBP1b during murein synthesis and suggest the existence of different peptidoglycan synthesis complexes.     

Role of the murein precursor UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala in repression of beta-lactamase induction in cell division mutants

Certain beta-lactam antibiotics induce the chromosomal ampC beta-lactamase of many gram-negative bacteria. The natural inducer, though not yet unequivocally identified, is a cell wall breakdown product which enters the cell via the AmpG permease component of the murein recycling pathway. Surprisingly, it has been reported that beta-lactamase is not induced by cefoxitin in the absence of FtsZ, which is required for cell division, or in the absence of penicillin-binding protein 2 (PBP2), which is required for cell elongation. Since these results remain unexplained, we examined an ftsZ mutant and other cell division mutants (ftsA, ftsQ, and ftsI) and a PBP2 mutant for induction of beta-lactamase. In all mutants, beta-lactamase was not induced by cefoxitin, which confirms the initial reports. The murein precursor, UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala (UDP-MurNAc-pentapeptide), has been shown to serve as a corepressor with AmpR to repress beta-lactamase expression in vitro. Our results suggest that beta-lactamase is not induced because the fts mutants contain a greatly increased amount of corepressor which the inducer cannot displace. In the PBP2(Ts) mutant, in addition to accumulation of corepressor, cell wall turnover and recycling were greatly reduced so that little or no inducer was available. Hence, in both cases, a high ratio of repressor to inducer presumably prevents induction.     

Characterization of an N-acetylmuramic acid/N-acetylglucosamine kinase of Clostridium acetobutylicum

We report here the cloning and characterization of a cytoplasmic kinase of Clostridium acetobutylicum, named MurK (for murein sugar kinase). The enzyme has a unique specificity for both amino sugars of the bacterial cell wall, N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc), which are phosphorylated at the 6-hydroxyl group. Kinetic analyses revealed Km values of 190 and 127 μM for MurNAc and GlcNAc, respectively, and a kcat value (65.0 s(-1)) that was 1.5-fold higher for the latter substrate. Neither the non-N-acetylated forms of the cell wall sugars, i.e., glucosamine and/or muramic acid, nor epimeric hexoses or 1,6-anhydro-MurNAc were substrates for the enzyme. MurK displays low overall amino acid sequence identity (24%) with human GlcNAc kinase and is the first characterized bacterial representative of the BcrAD/BadFG-like ATPase family. We propose a role of MurK in the recovery of muropeptides during cell wall rescue in C. acetobutylicum. The kinase was applied for high-sensitive detection of the amino sugars in cell wall preparations by radioactive phosphorylation.     

In vitro synthesis of cross-linked murein and its attachment to sacculi by PBP1A from Escherichia coli

The penicillin-binding protein (PBP) 1A is a major murein (peptidoglycan) synthase in Escherichia coli. The murein synthesis activity of PBP1A was studied in vitro with radioactive lipid II substrate. PBP1A produced murein glycan strands by transglycosylation and formed peptide cross-links by transpeptidation. Time course experiments revealed that PBP1A, unlike PBP1B, required the presence of polymerized glycan strands carrying monomeric peptides for cross-linking activity. PBP1A was capable of attaching nascent murein synthesized from radioactive lipid II to nonlabeled murein sacculi. The attachment of the new material occurred by transpeptidation reactions in which monomeric triand tetrapeptides in the sacculi were the acceptors.     

Low-molecular-weight substrate for the lysozyme of T4 bacteriophage.

It has been shown that muropeptide CB, the chemically defined product of Escherichia coli B murein digestion by phage lambda endolysin, is the substrate for T4 lysozyme. This is the tetrasaccharide GlcNAc-MurNAc-GlcNAc-anMurNAc in which the carboxyl groups of MurNAc and anMurNAc residues are substituted by tetrapeptide LAla-DGlu-msA2pm-DAla (MurNAc = N-acetylmuramic acid, GlcNAc = N-acetyl-D-glucosamine, anMurNAc = 1,6-anhydro-N-acetylmuramic acid, LAla = L-alanine, DGlu = D-glutamic acid, msA2pm = meso-diaminopimelic acid). The substrate contains one bond hydrolysable by T4 lysozyme. The products of hydrolysis are the easily identifiable disaccharide muropeptides C6 (GlcNAc-MurNAc-LAla-DGlu-msA2pm-DAla) and CA (GlcNAc-anMurNac-LAla-DGlu-msA2pm-DAla). Thus the substrate may be used for the specific identification of murein N-acetylmuramoylhydrolases of the T4 lysozyme type, as well as for any quantitative measurement of the enzymatic reaction.     

MurQ Etherase is required by Escherichia coli in order to metabolize anhydro-N-acetylmuramic acid obtained either from the environment or from its own cell wall

MurQ is an N-acetylmuramic acid-phosphate (MurNAc-P) etherase that converts MurNAc-P to N-acetylglucosamine-phosphate and is essential for growth on MurNAc as the sole source of carbon (T. Jaegar, M. Arsic, and C. Mayer, J. Biol. Chem. 280:30100-30106, 2005). Here we show that MurQ is the only MurNAc-P etherase in Escherichia coli and that MurQ and AnmK kinase are required for utilization of anhydro-MurNAc derived either from cell wall murein or imported from the medium.     

Cloning and characterization of PBP 1C, a third member of the multimodular class A penicillin-binding proteins of Escherichia coli.

All proteins of Escherichia coli that covalently bind penicillin have been cloned except for the penicillin-binding protein (PBP) 1C. For a detailed understanding of the mode of action of beta-lactam antibiotics, cloning of the gene encoding PBP1C was of major importance. Therefore, the structural gene was identified in the E. coli genomic lambda library of Kohara and subcloned, and PBP1C was characterized biochemically. PBP1C is a close homologue to the bifunctional transpeptidases/transglycosylases PBP1A and PBP1B and likewise shows murein polymerizing activity, which can be blocked by the transglycosylase inhibitor moenomycin. Covalently linked to activated Sepharose, PBP1C specifically retained PBP1B and the transpeptidases PBP2 and -3 in addition to the murein hydrolase MltA. The specific interaction with these proteins suggests that PBP1C is assembled into a multienzyme complex consisting of both murein polymerases and hydrolases. Overexpression of PBP1C does not support growth of a PBP1A(ts)/PBP1B double mutant at the restrictive temperature, and PBP1C does not bind to the same variety of penicillin derivatives as PBPs 1A and 1B. Deletion of PBP1C resulted in an altered mode of murein synthesis. It is suggested that PBP1C functions in vivo as a transglycosylase only.     

Two distinct transpeptidation reactions during murein synthesis in Escherichia coli.

Murein synthesized in ether-permeabilized cells of Escherichia coli deficient in individual penicillin-binding proteins (PBPs) and in the presence of certain beta-lactam antibiotics was analyzed by high-pressure liquid chromatography separation of the muramidase split products. PBP 1b was found to to be the major murein synthesizing activity that was poorly compensated for by PBP 1a. A PBP 2 mutant as well as mecillinam-inhibited cells showed increased activity in the formation of oligomeric muropeptides as well as UDP-muramylpeptidyl-linked muropeptides, the reaction products of transpeptidation, bypassing the lipid intermediate. In contrast, penicillin G and furazlocillin severely inhibited these reactions but stimulated normal dimer production. It is concluded that two distinct transpeptidases exist in E. coli: one, highly sensitive to penicillin G and furazlocillin, catalyzes the formation of hyper-cross-linked muropeptides, and a second one, quite resistant to these antibiotics, synthesizes muropeptide dimers.     

Physiological functions of D-alanine carboxypeptidases in Escherichia coli

Bacterial cell shape is, in part, mediated by the peptidoglycan (murein) sacculus. Penicillin-binding proteins (PBPs) catalyze the final stages of murein biogenesis and are the targets of beta-lactam antibiotics. Several low molecular mass PBPs including PBP4, PBP5, PBP6 and DacD seem to possess DD-carboxypeptidase (DD-CPase) activity, but these proteins are dispensable for survival in laboratory culture. The physiological functions of DD-CPases in vivo are unresolved and it is unclear why bacteria retain these seemingly non-essential and enzymatically redundant enzymes. However, PBP5 clearly contributes to maintenance of cell shape in some PBP mutant backgrounds. In this review, we focus on recent findings concerning the physiological functions of the DD-CPases in vivo, identify gaps in the current knowledge of these proteins and suggest some possible courses for future study that might help reconcile current models of bacterial cell morphology.     

Overproduction of inactive variants of the murein synthase PBP1B causes lysis in Escherichia coli

Penicillin-binding protein 1B (PBP1B) of Escherichia coli is a bifunctional murein synthase containing both a transpeptidase domain and a transglycosylase domain. The protein is present in three forms (alpha, beta, and gamma) which differ in the length of their N-terminal cytoplasmic region. Expression plasmids allowing the production of native PBP1B or of PBP1B variants with an inactive transpeptidase or transglycosylase domain or both were constructed. The inactive domains contained a single amino acid exchange in an essential active-site residue. Overproduction of the inactive PBP1B variants, but not of the active proteins, caused lysis of wild-type cells. The cells became tolerant to lysis by inactive PBP1B at a pH of 5.0, which is similar to the known tolerance for penicillin-induced lysis under acid pH conditions. Lysis was also reduced in mutant strains lacking several murein hydrolases. In particular, a strain devoid of activity of all known lytic transglycosylases was virtually tolerant, indicating that mainly the lytic transglycosylases are responsible for the observed lysis effect. A possible structural interaction between PBP1B and murein hydrolases in vivo by the formation of a multienzyme complex is discussed.